WO2016200063A1 - Procédé de détection de virus à l'aide de liposomes et d'un hybride liposome-polymère - Google Patents

Procédé de détection de virus à l'aide de liposomes et d'un hybride liposome-polymère Download PDF

Info

Publication number
WO2016200063A1
WO2016200063A1 PCT/KR2016/004956 KR2016004956W WO2016200063A1 WO 2016200063 A1 WO2016200063 A1 WO 2016200063A1 KR 2016004956 W KR2016004956 W KR 2016004956W WO 2016200063 A1 WO2016200063 A1 WO 2016200063A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
liposome
glycero
phosphatidylcholine
liposomes
Prior art date
Application number
PCT/KR2016/004956
Other languages
English (en)
Korean (ko)
Inventor
정봉현
정찬호
배판기
Original Assignee
재단법인 바이오나노헬스가드연구단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 재단법인 바이오나노헬스가드연구단 filed Critical 재단법인 바이오나노헬스가드연구단
Publication of WO2016200063A1 publication Critical patent/WO2016200063A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to a virus detection method using liposomes or liposome-polymer hybrids, and more particularly, to samples that are presumed to contain liposomes or liposome-polymer hybrids and viruses containing an electrochemically active substrate. After contacting, the present invention relates to a virus detection method comprising detecting the presence or absence of a virus through a change in current caused by the release of an electrochemically active substrate contained in a liposome or a liposome-polymer hybrid.
  • Liposomes are W / O / W type emulsions proposed by Bangham in the 1960s (J. Mol . Biol ., 13: 238, 1965), in which amphiphilic phospholipids are self-arranged by hydrophobic forces in the water phase.
  • the phospholipid membrane constituting the liposome has the same structure as the cell, and is easily used for cell introduction, and has a large space for supporting a water-soluble substance therein compared to other structures, so that it is used as a carrier and carrier for the water-soluble substance (Eur. J. Pharm . Biopham, 62: 110, 2006 ; Nat Rev. Drug Discov, 145:... 4, 1979).
  • liposomes are nano-sized (1 ⁇ m or less) capsules as phospholipid delivery and mediators, and they can contain both lipophilic and hydrophilic functional materials, so they are suitable for living organisms similar to human skin cells. It is a substance that, when added to a hydrophilic formulation, remains suspended and has surface stability.
  • Liposomes are spherical vesicles in which the phospholipid bilayer surrounds the water phase.
  • the lipid membrane is an amphiphilic phospholipid consisting of two hydrophobic fatty acid groups and a hydrophilic phosphate group, which forms a double membrane in aqueous solution, which forms closed vesicles like artificial cells.
  • the non-polar fatty acid tail faces the inside of the membrane and the polar head faces outward.
  • Incorporating drugs into such liposomes has been attracting attention as a structure of particle bodies prepared by assembling with polymers, drugs, and antigens, as it can enhance the therapy by reducing the toxicity of drugs and increasing their effects.
  • Liposomes are fully enclosed structures that include a lipid bilayer membrane containing encapsulated aqueous medium. Liposomes may comprise many concentric lipid bilayers (multilamellar vesicles or MLVs) or single membrane bilayers (unilamellar vesicles) separated by an aqueous phase.
  • the lipid bilayer consists of two lipid monolayers with hydrophobic "tail” and hydrophilic "head” regions. In the membrane bilayer, the hydrophobic “tails” in the lipid monolayer are arranged towards the center of the bilayer and the hydrophilic “heads” are arranged towards the aqueous phase.
  • the basic structure of liposomes can be prepared by known methods. For example, lipid molecules suspended in an organic solvent are evaporated under reduced pressure to form a dry film in a vessel, and an appropriate amount of aqueous phase is added to the vessel and the mixture is stirred. The mixture can then be prepared by standing without shaking for a time sufficient to form a multilamellar vesicle. Unilamella vesicles can also be prepared by known techniques (eg, US Pat.
  • Liposome-polymer hybrids are biofilm mimetic amphiphilic structures composed of low amounts of lipids (eg, phospholipids) and high molecular weight polymers (eg, amphiphilic block copolymers).
  • the liposome-polymer hybrid is composed of a lipid component having a biological function (receptor, molecular recognition, etc.) and a polymer having a structural function (structural stability, etc.) can be prepared to bind to the target material (Olubummo A1 et al. , Langmuir, 30 (1): .
  • Liposomes or liposome-polymer hybrids can be designed for diagnostic purposes.
  • Liposomes or liposome-polymer hybrids can be covalently bound to proteins, antibodies and immunoglobulins.
  • thiolated IgG or Protein A can be covalently bound to lipid vesicles and the thiolated antibody or Fab 'fragment can be bound to liposomes or liposome-polymer hybrids.
  • biosensor systems information is easier and more useful for analyzing data by detecting signals using the properties of nanomaterials that display color, fluorescence, or electromagnetic signals at the cellular or in vivo level.
  • Can provide Chemical and biosensors are materials or devices that detect and measure information from an object to be measured and change the measurable amount into a usable signal.
  • the sensor acquires information from the target, the sensor converts the signals into recognizable signals such as color, fluorescence, and electrical signals to assist human judgment.
  • the sensor recognizes the target material, it sends a signal through a signal converter for human recognition.
  • sensors used in biosensors require high selectivity and sensitivity to target materials to be detected.
  • Enzymes and antibodies have excellent substrate specificity and high binding capacity, but have the disadvantage of low stability and high price when immobilized in a sensor device.
  • Nanobiosensors are biosensors that are improved by advanced nanotechnology, which converts reactions by binding to biocognitive materials into signals, and refers to sensors that can quickly test specific materials. This is the same principle as the enzyme-substrate complex of the biological concept, in which one ligand is only reactive with one substance having a specific component for the ligand and measures the degree of reactivity.
  • Miniaturized biosensor using nanotechnology minimizes human injury and enables painless human diagnosis and has the advantage of directly analyzing single cells.
  • biosensors with improved operating characteristics such as high stability, fast response time, high sensitivity, and high selectivity using nanotechnology enable continuous measurement of human diagnosis and single-molecule analysis.
  • Detection of influenza viruses is most likely due to high specificity and sensitivity (0.01 to 100 pfu (plaque forming units) / mL) and assays based on reverse transcription polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription polymerase chain reaction
  • the disadvantage is the need for expensive equipment and reagents.
  • rapid influenza detection tests are mainly performed by LFIA (Lateral Flow Immunoassay), which can detect viruses using virus-specific antibodies.
  • RIDTs can only detect relatively high concentrations of virus (10 3 -10 4 pfu / mL) (Kwon, D. et al., J. Clin . Microbiol . 49: 437-438, 2011; Su, LC et al., Anal.
  • biosensors that can detect infectious agents at low concentrations (number of objects), have high reproducibility, and can be realized at low cost and miniaturization (Grieshaber, D.). et al., Sensors , 8: 1400-1458, 2008). Unlike conventional fluorescence-based sensors, the biosensor can detect a biological sample that is cloudy or shows autofluorescence using an electrochemical method without a pretreatment process using a small amount of sample.
  • amperometric sensors or impedimetric sensors using bioreceptors have been developed, they have low sensitivity (10 3 pfu / mL) and have difficulty in increasing the stability of functionalized electrodes with biosensors (Karerich-Pedersen, K. et al. , Biosens . Bioelectron . 49: 374-379, 2013; Caygill, RL et al., Anal. Chim. Acta ., 681: 8-15, 2010).
  • Korean Patent No. 1561395 treats a hemagglutinin-specific degrading enzyme to activate a virus and contacts the activated virus with amphiphilic particles under acidic conditions of pH 4-8.
  • a virus detection method for detecting the presence or absence of a virus by detecting a change in fluorescence intensity emitted by self-quenching dye present in sex particles by dequenching.
  • detection methods essentially include viral hemagglutinin degrading enzymes and require conditions for bringing the activated virus into contact with the amphiphilic particles under acidic conditions. Therefore, the additional use of a reagent such as hemagglutinin degrading enzyme is inexpensive in terms of cost, and the process of forming and maintaining conditions suitable for detection is complicated and inefficient.
  • the present inventors have made intensive efforts to develop a method for easily detecting a target virus in a short time.
  • the inventors have incorporated an electrochemically active substrate into liposomes or liposome-polymer hybrids and are specific to viruses. Preparing liposomes or liposome-polymer hybrids that bind to them, and then liposomes or liposome-polymer hybrids when the virus binds to its liposomes or liposome-polymer hybrids containing an electrochemically active substrate through its own lipid membrane or membrane protein. By releasing the embedded electrochemically active substrate, it was confirmed that the virus can be easily detected by showing a change in current, thereby completing the present invention.
  • the present invention includes an electrochemically active substrate, includes a liposome or a liposome-polymer hybrid, and when the virus binds to the liposome or the liposome-polymer hybrid, the liposome or Provided are a virus detecting composition and a kit for detecting a virus, wherein the electrochemically active substrate contained in the liposome-polymer hybrid exhibits a current change.
  • the present invention also includes the steps of (a) containing an electrochemically active substrate and contacting a liposome or a liposome-polymer hybrid with a virus-containing putative sample; And (b) it provides a virus detection method comprising the step of confirming the current change in accordance with the contact of (a).
  • ELMID Electroactive Liposome-Mediated Influenza Detection
  • FIG. 2 shows particle size analysis and polydispersity index of the liposomes for detecting viruses containing K 4 Fe (CN) 6 (Potassium ferrocyanide (II)) by dynamic light scattering (DLS). And zeta potential (Z potential).
  • CN CN 6
  • Potassium ferrocyanide (II) Dynamic light scattering
  • Z potential zeta potential
  • Figure 3 shows cryo-TEM (cryogenic transmission electron microscopy) images of influenza-responosive liposomes (scale bar: 100 nm).
  • FIG. 4 shows cryo-TEM images of 2009 pandemic H1N1 virus (A / california / 07/2009) bound to influenza-responosive liposomes (yellow dashed line: virus; scale bar: 100 nm).
  • Figure 5 shows the electrochemical reaction of the influenza-responsive liposomes before and after lyophilization, liposomes before and after freezing at 2009 pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL) and 37 °C After reaction for 30 minutes, it was measured by cyclic voltammetry (error bar: standard deviation of 3 independent experiments).
  • FIG. 6 is a cyclic voltammetry method for confirming the change in current generated when a sample containing influenza, a target virus, is added using an influenza-responsive liposome containing K 4 Fe (CN) 6 (Potassium ferrocyanide (II)).
  • CN CN
  • II Potassium ferrocyanide
  • Figure 7 shows the electrochemical reaction before and after the heat treatment of influenza-responsive liposomes, 2009 pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL) treated for 10 minutes at 100 °C to liposomes Peak currents when added (error bar: standard deviation of 3 independent experiments).
  • Figure 8 shows the sensitivity and specificity of the ELMID method of Figure 1,
  • (a) is a 2009 pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL) diluted at 10-fold intervals or Medium (control; gray) was added to the influenza-reactive liposomes, reacted at 37 ° C. for 30 minutes, and confirmed by cyclic voltammetry
  • (b) shows the peak current of (a) by virus concentration
  • ( c) is the addition of various species of influenza A and influenza B to the influenza-reactive liposomes, reacted at 37 ° C.
  • Figure 9 shows the detection of influenza virus contained in the NP (nasopharyngeal) swab sample using the ELMID method, (a) adding the NP swab sample to influenza-reactive liposomes, and lab-scale three-electrode system (glassy cyclic voltammetry using a carbon electrode, followed by peak current, and (b) shows an image of a screen-printed electrode (SPE) useful for point-of-care detection. c) shows that the NP swab sample was added to influenza-reactive liposomes, reacted at 35 ° C. for 30 minutes, and then the peak current was monitored on the SPE by cyclic voltammetry (error bar: 3 independent times). Standard deviation of the experiment).
  • SPE screen-printed electrode
  • the present invention relates to a virus detection method using a liposome or a liposome-polymer hybrid, and more particularly, a sample presumed to contain a liposome or a liposome-polymer hybrid and a virus containing an electrochemically active substrate. After contacting, the present invention relates to a virus detection composition, and a virus detection method using the same, wherein the presence or absence of a virus is detected through a change in current caused by the contact.
  • receptor is a component of the membrane, consisting of proteins, lipids, carbohydrates, and combinations thereof.
  • lipid refers to a compound soluble in organic solvents, such as fats, waxes, steroids, sterols, glycolipids, terpenes, fats. Fat-soluble vitamins, prostaglandins, carotene, and the like, but are not limited thereto.
  • sample may be from, but is not limited to, a biological or environmental source.
  • biological sources body fluids of animals, plants, microorganisms, May be obtained from tissues, gases, and may include plasma, serum, etc.
  • environmental sources it may include soil, water, crystals, food, industrial products, and the like.
  • the average diameter of liposomes containing K 4 Fe (CN) 6 prepared for the detection of viruses is 142.3 ⁇ 5.2nm (by DLS (Dynamic light) scattering), zeta potential of -58.96 ⁇ 3.9 mV, polydispersity index (PI) of 0.1047 ⁇ 0.09 (higher PI increases the aggregation of liposomes), and the number of ferrocyanide ions per liposome It was found to be 1.2 ⁇ 10 5 (see Example 1).
  • the influenza virus can be contacted and release of the embedded electrochemically active substrate without including an enzyme that degrades HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting the electrochemically active substrate, or various experimental conditions possible before the electrical signal detection It is sufficient to maintain the negative charge under, but not limited to.
  • virus detection according to virus concentration is possible, and even at a low virus concentration of 5.2 ⁇ 10 pfu / mL.
  • the virus was found to be detectable. It is 100 times more sensitive than virus influenza detection tests (RIDTs), and the measurement (diagnosis) time is about 30 minutes, similar to the existing method (see Example 4).
  • influenza-responsive liposomes specifically react to influenza virus, and RSV (Human respiratory syncytial virus) ) And no cross-reaction (see Example 4).
  • influenza virus contained in the nasopharyngeal swab sample by ELMID method as shown in Figure 9a ((i) lab-scale three-electrode system), the lowest value The lower the Ct value, the higher the virus concentration; the distribution of Ct values in the used NP swab sample ranges from 9.09 to 24.37, while the influenza negative shows the highest peak current. It appeared similar to the media negative control (media CTL) (see Example 5).
  • FIG. 9C ((ii) SPE, screen-printed electrode), a liposome: virus volume ratio of 7: 1 was used to increase the signal intensity, which was similar to the lab-scale three-electrode system. The result was obtained.
  • the present invention in one aspect, includes an electrochemically active substrate, comprises a liposome or a liposome-polymer hybrid, and when the virus binds to the liposome or the liposome-polymer hybrid, the liposome or liposome
  • the present invention relates to a virus detection composition and a detection kit, wherein the electrochemically active substrate contained in the polymer hybrid exhibits a current change while being released.
  • the virus may bind to liposomes or liposome-polymer hybrids through viral lipid membranes or membrane proteins.
  • the viral membrane protein may be characterized as being HA (hemagglutinin).
  • the lipid membrane may be characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), SM (Sphingomyelin).
  • the liposome-polymer hybrid is a biofilm-like amphiphilic structure composed of a low amount of lipids (eg, phospholipids) and high molecular weight polymers (eg, amphiphilic block copolymers).
  • lipids eg, phospholipids
  • high molecular weight polymers eg, amphiphilic block copolymers
  • the liposome-polymer hybrid may be made of a lipid component having a biological function (receptor, molecular recognition, etc.) and a polymer having a structural function (structural stability, etc.) and may be manufactured to bind to a target material (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem . Soc ., 81: 303-311, 1986; Binder WH et al., Angew Chem . 115 (47): 5980-6007, 2003; Binder WH et al., Angew Chem Int Ed Engl ., 42 (47): 5802-27, 2003).
  • the influenza virus can be contacted and release of the embedded electrochemically active substrate without including an enzyme that degrades HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting the electrochemically active substrate, or various experimental conditions possible before the electrical signal detection It is sufficient to maintain the negative charge under, but not limited to.
  • the electrochemically active substrate is K 4 Fe (CN) 6 (Potassium ferrocyanide (II)), Ascorbic acid (Ascorbic acid), Ru (NH 3 ) 6 Cl 3 (Hexaammineruthenium (III) chloride), Ferrocene, ferrocene derivatives, quinones, quinone derivatives, ruthenium ammine complexes, osmium (II), osmium (III), osmium (IV) complexes osmium complex, metallocene, metallocene derivatives, potassium hexa-cyanoferrate (II), Melola's blue, Prussian blue (Prussian blue) dichlorophenolindophenol (DCPIP), o-phenylenediamine (o-PDA), 3,4-dihydroxybenzaldehyde (3,4-hydroxybenzaldehyde 4-DHB)), viologen, 7,7,8,8-tetracyanoquinodimethan
  • the liposome or the liposome-polymer hybrid containing the electrochemically active substrate may be further characterized by containing the target receptor.
  • the target receptor may be characterized in that it binds to the lipid membrane or membrane protein of the virus.
  • the target receptor is GT1b (Ganglioside GT1b), GD1b (Ganglioside GD1b), GQ1b (Ganglioside GQ1b), phosphatidylcholine (Phosphatidylcholine), GM2 (Ganglioside GM2), GM1 (Ganglioside GM1), GD1D (Glio) GB3 (Ganglioside GB3), GB4 (Ganglioside GB4), Sphingolipid (3'-sulfogalactosyl-ceramide) and may be selected from the group consisting of cholesterol (cholesterol).
  • cholesterol cholesterol
  • the current change is cyclic voltammetry, square wave voltammetry, normal pulse voltammetry, differential pulse voltammetry. Or it may be characterized by checking by impedance (impedance).
  • the virus is influenza virus (influenza virus), rubella virus (rubella virus), varicella-zoster virus (varicella-zoster virus), HAV (hepatitis A), HBV (hepatitis B), HSV ( herpes simplex virus, poliovirus, small pox, human immunodeficiency virus, HIV, vaccinia virus, rabies virus, Epstein Barr virus, Leo It may be characterized in that it is selected from the group consisting of a virus (reovirus) and rhinovirus (rhinovirus).
  • the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmito
  • the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inos
  • PC
  • Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA 0 to HA 1 / HA 2 through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem ., 69: 531-569, 2000; White, J. et al., J. Cell. Biol ., 89: 674-679, 1981 ).
  • the influenza virus can be contacted to release the contained electrochemically active substrate without including an enzyme that degrades HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by the current change, for example, various experimental conditions possible to maintain the negative charge in the sample state for detecting the current change or before detecting the current change. It is sufficient to maintain the negative charge under, but not limited to.
  • the present invention provides a method comprising the steps of: (a) containing an electrochemically active substrate and contacting a liposome or a liposome-polymer hybrid with a virus-containing putative sample; And (b) relates to a virus detection method comprising the step of confirming the current change in accordance with the contact of (a).
  • the influenza virus can be contacted and release of the embedded electrochemically active substrate without including an enzyme that degrades HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting the electrochemically active substrate, or various experimental conditions possible before the electrical signal detection It is sufficient to maintain the negative charge under, but not limited to.
  • the electrochemically active substrate is K 4 Fe (CN) 6 (Potassium ferrocyanide (II)), Ascorbic acid (Ascorbic acid), Ru (NH3) 6Cl3 (Hexaammineruthenium (III) chloride), Ferrocene (ferrocene) ), Ferrocene derivatives, quinones, quinone derivatives, ruthenium ammine complexes, osmium (II), osmium (III), osmium (IV) complex , Metallocene, metallocene derivatives, potassium hexa-cyanoferrate (II), Melola's blue, Prussian blue Dichlorophenolindophenol (DCPIP), o-phenylenediamine (o-PDA), 3,4-dihydroxybenzaldehyde (3,4-DHB) )), Viologen, 7,7,8,8-tetracyanoquinodimethane (7,7,8,8-tetracyanoquino
  • the liposome or the liposome-polymer hybrid containing the electrochemically active substrate may be further characterized by containing the target receptor.
  • the target receptor may be characterized in that it binds to the lipid membrane or membrane protein of the virus.
  • the viral membrane protein may be characterized as being HA (hemagglutinin).
  • the lipid membrane may be characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), SM (Sphingomyelin).
  • the target receptor is GT1b (Ganglioside GT1b), GD1b (Ganglioside GD1b), GQ1b (Ganglioside GQ1b), phosphatidylcholine (Phosphatidylcholine), GM2 (Ganglioside GM2), GM1 (Ganglioside GM1), GD1D (Glio) GB3 (Ganglioside GB3), GB4 (Ganglioside GB4), Sphingolipid (3'-sulfogalactosyl-ceramide) and may be selected from the group consisting of cholesterol (cholesterol).
  • cholesterol cholesterol
  • the current change of step (b) indicates that when the virus binds to the liposome or liposome-polymer hybrid through the lipid membrane or membrane protein, the electrochemically active substrate contained in the liposome is released to show the current change. It can be characterized.
  • checking whether the current changes in the step (b) may be performed through an oxidation or reduction reaction.
  • the current change is cyclic voltammetry, square wave voltammetry, normal pulse voltammetry, differential pulse voltammetry. Or it may be characterized by checking by impedance (impedance).
  • the virus is influenza virus (influenza virus), rubella virus (rubella virus), varicella-zoster virus (varicella-zoster virus), HAV (hepatitis A), HBV (hepatitis B), HSV ( herpes simplex virus, poliovirus, small pox, human immunodeficiency virus, HIV, vaccinia virus, rabies virus, Epstein Barr virus, Leo It may be characterized in that it is selected from the group consisting of a virus (reovirus) and rhinovirus (rhinovirus).
  • the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmito
  • the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inos
  • PC
  • Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA 0 to HA 1 / HA 2 through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem . , 69: 531-569, 2000; White, J. et al., J. Cell. Biol. , 89: 674-679, 1981 ).
  • the influenza virus can be contacted to release the contained electrochemically active substrate without including an enzyme that degrades HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by the current change, for example, various experimental conditions possible to maintain the negative charge in the sample state for detecting the current change or before detecting the current change. It is sufficient to maintain the negative charge under, but not limited to.
  • liposomes or liposome-polymer hybrids may contain (capture) various molecules such as absorbers, fluorescent materials, electrochemicals, or chemiluminescent materials in the inner aqueous phase. It is possible to change the composition of liposomes and the ability to capture a large number of substances, it is possible to obtain amplified signals and instantaneous signal can detect a small amount of virus in a short time.
  • Example 1 Preparation of liposomes or liposome-polymer hybrids containing K 4 Fe (CN) 6 (Potassium ferrocyanide (II))
  • For virus detection liposomes 80 mol% POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids Inc., USA), 20 mol% POPG (1-palmitoyl-2-oleoyl-sn-glycero Dissolved in chloroform using -3-phospho- (1'-rac-glycero), Avanti Polar Lipids Inc., USA), and then evaporated the chloroform for at least 1 hour under reduced pressure, and the chloroform was evaporated. Drying in a vacuum oven for 1 day to form a thin lipid film.
  • Liposome suspensions were prepared by dispersing 0-50 ⁇ M K 4 Fe (CN) 6 (Potassium ferrocyanide (II)) (Sigma-Aldrich, USA) for 10 minutes with an ultrasonicator (Jeiotech, Korea). Here, the process of freezing and thawing for the preparation of liposomes of a single lipid layer was repeated five times.
  • the liposomes of the same size were prepared after passing through a 100-200 nm pore size filter using a Mini-Extruder (Avanti Polar Lipids Inc., USA) for the production of uniform size liposomes.
  • liposomes containing POPC / POPG (molar ratio 4: 1) prepared as liposomes for virus detection were named influenza-responsive liposomes.
  • Figure 3 shows the cryo-TEM (cryogenic transmission electron microscopy) image of influenza-responsive liposomes.
  • a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on a platinum electrode, applying an electric field, and adding distilled water.
  • liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl . , 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem .
  • the liposome is a lipid receptor (Ganglioside GT1b), GD1b (Ganglioside GD1b), GQ1b (Ganglioside GQ1b), Phosphatidylcholine (GM2), GM2 (Ganglioside GM2), Specific to GM1 (Ganglioside GM1), GD1a (Ganglioside GD1a), GB3 (Ganglioside GB3), GB4 (Ganglioside GB4), Sphingolipid (3'-sulfogalactosyl-ceramide), Cholesterol, or Virus Binding proteins, lipids and glycosylation moieties were prepared.
  • the average diameter of liposomes containing K 4 Fe (CN ) 6 prepared for the detection of viruses is 142.3 ⁇ 5.2nm (by DLS (Dynamic light scattering), Zeta Potential) Silver -58.96 ⁇ 3.9mV, polydispersity index (PI) was 0.1047 ⁇ 0.09 (higher PI increased the aggregation of liposomes), and the number of ferrocyanide ions per liposome was 1.2 ⁇ 10 5 appear.
  • Table 1 shows the results of predicting the number of K 4 Fe (CN) 6 (Potassium ferrocyanide (II)) contained in the virus detection liposomes.
  • C Fe is determined by comparing the standard ferrocyanide concentration and the standard curve of peak current.
  • Influenza viruses prepared by conventional methods in the art are treated to liposomes containing K 4 Fe (CN) 6 (Potassium ferrocyanide (II)) at an appropriate concentration and exposure time and subjected to cyclic voltammetry [Fe ( CN) 6 ] 4- (ferrocyanide) release (degree) was measured to detect the virus.
  • a method of detecting influenza virus using liposomes containing ferrocyanide was named ELMID (Electroactive Liposome-Mediated Influenza Detection) (see FIG. 1).
  • liposomes encompassing the K 4 Fe (CN) 6 is liposomes encompassing K 4 Fe (CN) 6 - may be replaced by the polymer hybrid.
  • a buffer citrate-phosphate buffer, pH 5.0
  • Liposomes containing K 4 Fe (CN) 6 may be prepared to further contain virus specific membrane proteins, lipid membranes or glycosylation moieties.
  • the electrochemical cell is then filled with a sample containing the liposomes and virus, and the potential is set by bringing three electrodes (glassy carbon, reference electrode (Ag / AgCl) and counter electrode (Pt)) into contact with the solution.
  • the oxidation and reduction currents were measured by cyclic voltammetry (CV) by adjusting (eg, -0.2 V and 0.6 V at 100 mVs -1 vs. Ag / AgCl).
  • CV cyclic voltammetry
  • the differential pulse current curve can be obtained by applying a voltage in a suitable range to the electrochemically active substrate to the working electrode through a constant potential.
  • pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL; red) or medium (control; gray) was added to the influenza-reactive liposomes, followed by 30 min reaction at 37 ° C., followed by circulating voltage Amperometric confirmation of the release of ferrocyanide (degree).
  • pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL; red) or medium (control; grey) is added to the influenza-responsive liposomes and then reacted at 37 ° C. for 0-60 minutes, The release of ferrocyanide was confirmed by cyclic voltammetry at 5 minute intervals.
  • Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA 0 to HA 1 / HA 2 through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem ., 69: 531-569, 2000; White, J. et al., J. Cell. Biol. , 89: 674-679, 1981 ).
  • Liposomes with negative charges have been reported to bind and fuse with viruses even at low or neutral pH (Haywood, AM et al., Proc . Natl . Acad . Sci. USA , 82: 4611-4615, 1985; Nussbaum, O. et al., J. Gen. Virol . 73: 2831-2837, 1992). Therefore, in order to confirm the release of pH-dependent ferrocyanide from the negatively charged influenza-responsive liposome prepared in Example 1, the reaction was performed at 37 ° C.
  • pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL) was added 20 ⁇ L of TPCK-trypsin (Thermo Fisher Scientific, 0.1 unit / ⁇ L) and treated at 37 ° C. for 30 minutes. . Then, trypsin-treated 2009 pandemic H1N1 virus was added to liposomes, and reacted at 37 ° C. for 30 minutes, and then the release of ferrocyanide was confirmed by cyclic voltammetry.
  • TPCK-trypsin Thermo Fisher Scientific, 0.1 unit / ⁇ L
  • the virus membrane protein HA hemagglutinin
  • the virus was heated and reacted with liposomes. That is, 2009 pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL) (control: unheat-treated virus) treated for 10 minutes at 100 ° C. was added to liposomes to inactivate the HA of the virus. The peak currents measured by cyclic voltammetry were compared (see Geiss, GK et al., J. Virol . 75: 4321-4331, 2001).
  • the ELMID method was able to detect the virus independent of the HA activity of the virus when measuring the binding between the virus and liposomes by cyclic voltammetry.
  • liposomes encompassing the K 4 Fe (CN) 6 is liposomes encompassing K 4 Fe (CN) 6 - may be replaced by the polymer hybrid.
  • Lyophilization of influenza-responsive liposomes is accomplished using Freeze drier (FDU-2100, EYELA, Japan), (i) freezing at -80 ° C for 2 hours, and (ii) vacuum at -80 ° C overnight. After drying in the state, (iii) secondary drying overnight at 25 ° C. When hydrating lyophilized liposomes, lyophilized liposomes were added to the same volume of distilled water and shaken carefully to obtain hydrated liposomes.
  • Freeze drier FDU-2100, EYELA, Japan
  • virus titer The detection of virus according to virus type (influenza A, influenza B, RSV A and RSV B) and virus titer was compared by cyclic voltammetry.
  • the liposome containing K 4 Fe (CN) 6 may be replaced with a liposome-polymer hybrid containing K 4 Fe (CN) 6 .
  • Sensitivity 2009 pandemic H1N1 virus (A / california / 07/2009, 5.2 ⁇ 10 6 pfu / mL) diluted in 10-fold intervals, medium (Media CTL, negative control) or Triton X at appropriate concentration -100 (positive control) was added to 100 mM KCl and 100 mM CP buffer (citrate-phosphate buffer, pH 5.0) containing influenza-responsive liposomes, reacted at 37 ° C for 30 minutes, and then detected by cyclic voltammetry. It was confirmed.
  • the virus can be detected according to the virus concentration (titer), and the virus can be detected even at a low virus concentration of 5.2 ⁇ 10 pfu / mL.
  • This virus was 100 times more sensitive than conventional rapid influenza detection tests (RIDTs), and the measurement (diagnosis) took about 30 minutes.
  • the viral load contained in the nasal swab or throat swab of a typical influenza infected patient is 10 to 10 4 pfu / mL (Suess, T. et al. PLoS One 7: e51653, 2012).
  • the ELMID method is very useful for virus detection.
  • the virus can be detected using the cyclic voltammetry regardless of influenza virus species. That is, the ELMID method is a very useful virus detection method in that the virus can be detected irrespective of the mutant type of the viral membrane protein expressed in the newly appearing mutant influenza virus.
  • ELMID Electroactive Liposome-Mediated Influenza Detection
  • RSV Human
  • ELMID detection of RSV Weissenhorn, W. et al., FEBS Lett ., 581: 2150-2155, 2007; Bawage, SS et al., Adv . Virol . , 2013: 595768, 2013; Chaiwatpongsakorn, S. et al., J. Virol ., 85: 3968-3977, 2011).
  • influenza-responsive liposomes were found to specifically react to influenza virus and not cross-react with RSV.
  • Table 2 shows the electrochemical reactions of 14 influenza viruses using the ELMID method.
  • liposomes containing K 4 Fe (CN) 6 used for virus detection can be replaced with liposome-polymer hybrids containing K 4 Fe (CN) 6 .
  • FIG. 9C SPE, screen-printed electrode
  • a liposome: virus volume ratio of 7: 1 was used to increase the signal intensity. Similar results to the lab-scale three-electrode system were obtained. .
  • parainfluenza parainfluenza type 3 virus was added to influenza-reactive liposomes, and then reacted at 37 ° C. for 30 minutes, and then the release (degree) of K 4 Fe (CN) 6 was confirmed by cyclic voltammetry.
  • Each control group 'Media CTRL' means no virus
  • 'HPIV3' means parainfluenza type 3 virus (6 ⁇ 10 4 pfu / ml)
  • '' influenza 'means 2009 pandemic H1N1 virus (A / california / 07/2009, meaning 2.72 ⁇ 10 5 pfu / mL).
  • influenza-responsive liposomes were confirmed to specifically react to influenza virus and not cross-react with parainfluenza type 3.
  • MDCK Mesh-Darby Canine Kidney, ATCC CCL-344
  • ATCC American Type Culture Collection
  • MDCK cells 10% FBS ( cultured in MEM medium (Minimal Essential Medium) (Gibco BRL, Grand Island, USA) containing fetal bovine serum), 1x antibiotic-antimycotic mixture, 1x MEM vitamin solution, and 50 ⁇ g / ml gentamicin.
  • FBS cultured in MEM medium (Minimal Essential Medium) (Gibco BRL, Grand Island, USA) containing fetal bovine serum
  • 1x antibiotic-antimycotic mixture 1x MEM vitamin solution
  • 50 ⁇ g / ml gentamicin 50 ⁇ g / ml gentamicin.
  • Nasopharyngeal (NP) samples were collected from seven patients with symptoms of influenza virus, and the samples were added to virus transport media (UTM, Copan Diagnostics Inc., USA) and stored at -70 ° C.
  • virus transport media ULM, Copan Diagnostics Inc., USA
  • the AdvanSure RV real-time PCR kit LG Life Sciences, Korea
  • the Ct (threshold cycle) value was obtained according to the instruction manual of the SLAN Real-Time Quantitative PCR Detection System.
  • a positive response to influenza virus was determined by using a cut-off of Ct of 25.
  • Virus detection method using a liposome or liposome-polymer hybrid containing an electrochemically active substrate according to the present invention is not electrochemically active substrate out of the liposome or liposome-polymer hybrid, high stability to oxygen or chemical reaction, When the virus binds to liposomes or liposome-polymer hybrids, the virus detection signal is markedly high because the electrochemically active substrate contained in the liposomes or liposome-polymer hybrids reacts with the electroinductive substance to show a change in current.

Abstract

La présente invention concerne un procédé de détection de virus à l'aide de liposomes ou d'un hybride liposome-polymère, et plus particulièrement, un procédé de détection de virus dans lequel un échantillon contenant vraisemblablement un virus est mis en contact avec des liposomes ou un hybride liposome-polymère contenant une matrice active électrochimique, puis la présence ou l'absence d'un virus est détectée au moyen d'un changement de courant dû à l'émission de la matrice active électrochimique contenue dans les liposomes ou l'hybride liposome-polymère due au contact. Dans un procédé de détection de virus à l'aide de liposomes ou d'un hybride liposome-polymère contenant une matrice active électrochimique, lorsqu'un virus se lie à des liposomes ou à un hybride liposome-polymère, une matrice active électrochimique contenue dans les liposomes ou dans l'hybride liposome-polymère est émise puis un changement de courant se produit et, de ce fait, un signal de détection de virus s'avère considérablement élevé.
PCT/KR2016/004956 2015-06-11 2016-05-12 Procédé de détection de virus à l'aide de liposomes et d'un hybride liposome-polymère WO2016200063A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20150082788 2015-06-11
KR10-2015-0082788 2015-06-11
KR10-2016-0056901 2016-05-10
KR20160056901 2016-05-10

Publications (1)

Publication Number Publication Date
WO2016200063A1 true WO2016200063A1 (fr) 2016-12-15

Family

ID=57503346

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2016/004956 WO2016200063A1 (fr) 2015-06-11 2016-05-12 Procédé de détection de virus à l'aide de liposomes et d'un hybride liposome-polymère

Country Status (1)

Country Link
WO (1) WO2016200063A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR980010417A (ko) * 1996-07-13 1998-04-30 구자홍 전기화학식 면역 바이오센서
KR100762202B1 (ko) * 2005-10-18 2007-10-04 건국대학교 산학협력단 용혈성 미생물을 선택적으로 탐지하는 방법
US7829272B2 (en) * 2007-05-24 2010-11-09 Nanotrope Inc. Viral detection liposomes and method
JP5047783B2 (ja) * 2004-05-13 2012-10-10 サントル・ナシオナル・ドゥ・ラ・ルシェルシュ・シアンティフィーク（セーエヌエールエス） ターゲット存在物をおとり存在物へ結合させるための装置、および該装置を使用する検出方法
JP2013061325A (ja) * 2011-08-22 2013-04-04 Liposome Engineering Laboratory リポソームを用いた酵素免疫測定技術ＬＥＬＩＡ（Ｌｉｐｏｓｏｍｅ−ｂａｓｅｄＥｎｚｙｍｅ−ＬｉｎｋｅｄＩｍｍｕｎｏＡｓｓａｙ）

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR980010417A (ko) * 1996-07-13 1998-04-30 구자홍 전기화학식 면역 바이오센서
JP5047783B2 (ja) * 2004-05-13 2012-10-10 サントル・ナシオナル・ドゥ・ラ・ルシェルシュ・シアンティフィーク（セーエヌエールエス） ターゲット存在物をおとり存在物へ結合させるための装置、および該装置を使用する検出方法
KR100762202B1 (ko) * 2005-10-18 2007-10-04 건국대학교 산학협력단 용혈성 미생물을 선택적으로 탐지하는 방법
US7829272B2 (en) * 2007-05-24 2010-11-09 Nanotrope Inc. Viral detection liposomes and method
JP2013061325A (ja) * 2011-08-22 2013-04-04 Liposome Engineering Laboratory リポソームを用いた酵素免疫測定技術ＬＥＬＩＡ（Ｌｉｐｏｓｏｍｅ−ｂａｓｅｄＥｎｚｙｍｅ−ＬｉｎｋｅｄＩｍｍｕｎｏＡｓｓａｙ）

Similar Documents

Publication Publication Date Title
Zhuang et al. Liposome-amplified photoelectrochemical immunoassay for highly sensitive monitoring of disease biomarkers based on a split-type strategy
KR101814495B1 (ko) 바이러스 검출용 키트
Connor et al. pH-sensitive immunoliposomes as an efficient and target-specific carrier for antitumor drugs
Schoen et al. Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles
Qu et al. An electrochemical immunosensor based on enzyme-encapsulated liposomes and biocatalytic metal deposition
CN111500284B (zh) 一种包封石墨烯量子点的纳米脂质体及制备以及其在生物酶活性检测中的应用
Kubesch et al. Interaction of polymyxin B nonapeptide with anionic phospholipids
Razinkov et al. RSV entry inhibitors block F-protein mediated fusion with model membranes
US9494524B2 (en) Assay for analytes based on aggregation
WO2016200063A1 (fr) Procédé de détection de virus à l'aide de liposomes et d'un hybride liposome-polymère
KR101755408B1 (ko) 리포좀 또는 리포좀 폴리머 하이브리드를 이용한 박테리아 검출방법
Ballesteros et al. Autophagy protein LC3C binding to phospholipid and interaction with lipid membranes
Yoshida et al. Simple preparation and characterization of cationic liposomes associated with a monoclonal antibody against glioma-associated antigen (immunoliposomes)
WO2016137236A1 (fr) Procédé de détection de virus utilisant un liposome ou un hybride liposome-polymère
Kozuma et al. Fusion characteristics of a nuclear polyhedrosis virus in cultured cells: time course and effect of a synergistic factor and pH
Muñoz-Barroso et al. Dynamic properties of Newcastle Disease Virus envelope and their relations with viral hemagglutinin-neuraminidase membrane glycoprotein
Faggiano et al. Phospholipid components of the synthetic pulmonary surfactant CHF5633 probed by fluorescence spectroscopy
WO2016200064A1 (fr) Procédé de détection de bactéries à l'aide de liposomes et d'un hybride liposome-polymère
WO2016111568A1 (fr) Kit de détection de virus
KR20180090513A (ko) 바이러스 검출용 키트
EP0258388A1 (fr) Analyse immunologique de liposomes - procedes et produits
MacDonald Energy transfer measurements of fusion between Sendai virus and vesicles corrected for decreased absorption of acceptor probe.
WO2016111572A1 (fr) Nécessaire de détection de virus
Groß et al. Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens
Xu Towards the Development of Organic Electrochemical Devices for Enveloped Virus Detection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16807704

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16807704

Country of ref document: EP

Kind code of ref document: A1